Catalase-(EC 1.11.1.6) is a tetrameric enzyme with a total molecular weight (MW) of about 323,000 daltons (d). It is capable of reducing hydrogen peroxide to water and molecular oxygen in the stochiometric reaction. ##STR1##
The decomposition of hydrogen peroxide proceeds by a two step reaction catalysed by a heme iron complex that serves as the active site of the enzyme.
Catalase's characteristic activity of decomposing hydrogen peroxide makes it a valuable component in a number of processes. For example, a process for the production of purified oil seed protein according to U.S. Pat. No. 4,464,296 requires treating oil seed protein with sufficient peroxide to increase the solubility of the protein. The solubilized protein is collected, dialysed against water containing catalase, and the dialysate is freeze dried to yield dry purified oil seed protein.
U.S. Pat. No. 4,460,686 describes the oxidation of glucose using an immobilized glucose oxidase-catalase composition in a reaction mixture at temperatures of 1.degree.-2.degree. C. Catalase activity in the process is maintained at least 1/6th of the glucose oxidase activity in the composition. Long reaction life is maintained by running the oxidation at low temperature.
As described in U.S. Pat. No. 4,101,581 catalase is used in a method for determining the presence of substances in fluids, particularly, biological fluids, that form hydrogen peroxide. Catalase and methanol are used to produce formaldehyde from peroxide. The formaldehyde reacts with a hydrozone in the presence of ferric chloride to form a dye that can be determined photometrically.
Catalase and glucose oxidase bound to an appropriate carrier in immediate proximity to one another are used to convert glucose to gluconic acid according to a method described in U.S. Pat. No. 3,935,071. Peroxide produced by the glucose oxide-mediated oxidation of glucose to gluconic acid is convered to water and molecular oxygen by the bound catalase. The coimmobilization of the two enzymes extends the catalyst activity according to the patent and minimizes the inactivation of glucose oxidase by peroxide.
Catalase is also used to convert hydrogen peroxide to water and molecular oxygen in a process for the production of fructose from glucose via the intermediate glucosone, as described in U.S. Pat. Nos. 4,246,347 and 4,423,149. In these patents glucose is reacted with enzymes capable of converting the hydroxyl group at the two position of glucose to a carbonyl in the presence of oxygen. Enzymes capable of carrying out this specific conversion include pyranose-2-oxidase (P-2-0) and glucose-2-oxidases (G-2-0s). Hydrogen peroxide, produced as one product of the enzymatic reaction mediated by P-2-0, oxidizes certain critical sites on the P-2-0 enzyme molecule, damaging its function. Catalase is added to the reaction solution to remove the hydrogen peroxide. The process described in these patents can be conducted within a temperature range from about 15.degree. C. to about 65.degree. C.
Catalase is also used in a process for enhancing the properties of tobacco as described in U.S. Pat. No. 3,889,689. In this process catalase and a liquid containing hydrogen peroxide is forced to permeate the interstices of tobacco where the catalase and hydrogen peroxide react in situ.
A positive image photographic process which uses layers containing catalase is described in U.S. Pat. No. 3,694,207. In this process catalase reacts with hydrogen peroxide to form an image of gas bubbles in the layer, or to produce a dye image by a color-forming oxidation reaction. The catalase is inactivated upon exposure to light.
From the foregoing, it is clear that the enzyme catalase is used in numerous process in which the activity of the enzyme must be maintained without inactivation for at least some period of time during the process. The irreversible dissociation of enzymes into subunits is known to inactivate enzymes. Catalase, a four subunit enzyme is certainly inactivated by dissociation into subunits. The intramolecular crosslinking of enzymes by bifunctional crosslinking reagents is an important tool in the field of enzyme immobilization and stabilization against inactivation.
The effect of crosslinking on the activity and the characteristics of an enzyme, is difficult if not impossible to predict. Crosslinking of one enzyme may yield activity enhancement while crosslinking of a second enzyme may yield no enhancement or even loss of activity. In fact, crosslinking may even cause increased activity and decreased activity in a single bifunctional enzyme. For example, bovine pancreatic ribonuclease A was crosslinked with the bifunctional di-imido ester dimethyl adipimidate. The resulting crosslinked monomeric enzyme displayed an increase in specific activity toward cytidine 2',3'-cyclic phosphate and a decrease in activity toward RNA. Hartman, F. C. and Wold, F. Cross-linking of Bovine Pancreatic Ribonuclease A with Dimethyl Adipimidate, Biochemistry, 6(8):2439-2448 (1967).
Thermal inactivation of enzymes may take place at elevated temperatures. By the term elevated temperatures is meant temperatures significantly higher than the temperature that is normally ambient for the organism from which the enzyme was obtained. Thermal inactivation is an important phenomenon in industrial enzymatic processes for a number of reasons.
Chemical and enzymatic reaction rates generally accelerate as temperature increases. If thermal inactivation is prevented, a temperature increase from 25.degree.-70.degree. C. will yield a 100-fold increase in the reaction rate. Thus, from the standpoint of process economics, the use of high temperatures in commercial enzymatic processes is advantageous.
The probability of bacterial contamination is reduced in enzyme reactors run at high temperatures. The deleterious effects of such bacterial contamination are many and include, for example, the liberation of enzyme degrading proteases, the plugging of filters, the production of unwanted by-products and increased cost of the process cycle. Because of the severity of this problem in the food industry, most enzymatic food processes are carried out at temperatures in excess of 60.degree. C.
Process productivity may be increased by maximizing the concentration of dissolved substate in an enzyme reactor. The soluability of most substrates increases with temperature. For example, starch, a polymer of glucose, is gelatinized at temperatures between 100.degree.-110.degree. C.
Examples of industrial processes carrying out elevated temperatures include the production of high-fructose syrup from glucose using glucose isomerase, and alpha amylase and glucoamylase-catalysed hydrolysis of starch.